WO2002068686A1 - Methods for analysis of rna - Google Patents
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- WO2002068686A1 WO2002068686A1 PCT/GB2002/000868 GB0200868W WO02068686A1 WO 2002068686 A1 WO2002068686 A1 WO 2002068686A1 GB 0200868 W GB0200868 W GB 0200868W WO 02068686 A1 WO02068686 A1 WO 02068686A1
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- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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- the invention relates to the detection and analysis of RNA transcripts and in particular to methods of characterising the numbers and types of primary RNA transcripts produced in a given cell or tissue.
- the present invention now provides a method which enables analysis of the missing level in the hierarchy of information transfer, namely the numbers and types of nascent transcripts (i.e. those still associated with engaged polymerases) .
- the method is a general technique for quantitative cataloguing of both genie and non-genic transcripts in one or more cell populations.
- Useful applications of the technique include monitoring of the changes that occur when cells differentiate, or become malignant, or when they are exposed to exogenous agents (e.g. viruses, chemicals, carcinogens, ⁇ -rays, UV light) . Comparisons of different transcript profiles (e.g. of cancer cells and their normal precursors) should allow identification of which transcription units have been repressed or activated (e.g. during tumorigenesis) , and in turn this should lead to the development of diagnostic probes, and to the identification of possible targets for therapeutic intervention.
- Various high-throughput methods including those based on SAGE (Velculescu et al . , 1995; Kinzler et al . , US Patent No.
- RNA selected for analysis contains a poly (A) tail, and so does not contain primary transcripts. This bias results because the poly (A) + transcripts analysed are necessarily genie transcripts, and because non-genic targets are not usually placed on the microarrays or in SAGE databases.
- RNA transcripts transcribed in the cell or cell population with a nucieotide analogue
- purifying the labelled transcripts synthesising first strand cDNA copies of the labelled transcripts
- the invention further relates to a method for analysing a population of RNA transcripts which comprises:
- RNA transcripts ligating the 3' ends of the RNA transcripts to single-stranded oligonucleotide linkers comprising an enzyme recognition site that allows DNA cleavage at a site spaced a defined distance from the recognition site to form linked RNA;
- the invention further provides a nucleic acid composition derived from a cell or cell population comprising at least one ditag, wherein each ditag comprises two covalently joined nucleic acid tags in opposite orientation, wherein each tag corresponds to the extreme 3 1 end of a primary RNA transcript expressed in said cell or cell population.
- the invention further provides a method of detecting and characterising transcripts associated with a specific transcription unit within a pool of RNA, the method comprising steps of:
- the invention relates to the detection and analysis of RNA transcripts produced in a cell or cell population by means of the following steps :
- step (iv) comprises analysing the resultant cDNA to determine the nature of the primary RNA transcripts produced in the cell or cell population.
- step (i) comprises analysing the resultant cDNA to determine the nature of the primary RNA transcripts produced in the cell or cell population.
- nascent RNA transcripts transcribed in the cell or cell population are labelled with a nucieotide analogue in step (i).
- RNA processing including mature mRNAs . It is known to label nascent RNA transcripts within a cell with a nucieotide analogue by allowing controlled extension of RNA transcripts actively engaged to an RNA polymerase in the presence of a labelled nucieotide analogue (Iborra et al . , 1996 and 1998; Jackson et al . , 1998).
- nascent RNA may be separated from total cellular RNA using an antibody specific for the nucieotide analogue, whilst nascent RNA labelled with a biotinylated nucieotide analogue may be purified using streptavidin-magnetic beads.
- RNA transcripts incorporating labelled nucieotide analogues are capable of being faithfully copied by the enzyme reverse transcriptase to form a cDNA.
- the finding that transcripts incorporating labelled analogue nucleotides can be copied into cDNA has led to the possibility of using the powerful techniques available for high-throughput analysis of cDNA populations in order to analyse populations of nascent RNA transcripts.
- the method of the invention is critically dependent on labelling of RNA transcripts.
- labelling of nascent RNA molecules allows them to be purified from total cellular RNA.
- Labelling is accomplished by incorporation of a nucieotide analogue into the nascent transcripts.
- the nucieotide analogue may be essentially any nucieotide analogue having the following properties: 1) capable of being incorporated into an RNA strand, 2) permits selection of labelled versus non-labelled transcripts, and
- Suitable nucieotide analogues include, but are not necessarily limited to, analogues which capable of being recognised by specific antibodies (e.g. Br-UTP) , analogues such as biotin-CTP which can be recognised via a high specificity binding reaction (i.e. biotin/avidin or biotin/streptavidin binding) and also analogues which are directly detectable as a result of some property, such as fluorescence, luminescence etc, for example fluorescein-UTP.
- specific antibodies e.g. Br-UTP
- analogues such as biotin-CTP which can be recognised via a high specificity binding reaction (i.e. biotin/avidin or biotin/streptavidin binding)
- analogues which are directly detectable as a result of some property such as fluorescence, luminescence etc, for example fluorescein-UTP.
- Labelling of nascent transcripts may be accomplished using techniques known in the art. Suitable approaches are described, for example, by
- RNA RNA containing the nucieotide analogue, so that the analogue is incorporated into nascent RNA.
- the resulting labelled RNA can then be purified free of non-labelled (e.g. completed) transcripts, for example using an antibody that reacts specifically with the labelled RNA.
- a different approach involves permeabilizing cells with saponin, and allowing still-engaged polymerases to extend nascent transcripts in the presence of the labelled nucieotide, before labelled RNA is selected as before. The inventors have observed in control experiments that:
- nucieotide analogues are incorporated into RNA (using RNAse and inhibitors of the different RNA polymerases),
- cells are most preferably permeabilized and allowed to extend nascent transcripts by -25 nucleotides in the nucieotide analogue (See Jackson et al. (1998) for methods for achieving and monitoring such extensions) .
- Labelled RNA transcripts are purified, i.e. selected from the pool of total cellular RNA, and then converted to first strand cDNA.
- first strand cDNA synthesis may be facilitated by ligating single-stranded oligonucleotide linkers to the 3' ends of the labelled transcripts.
- First-strand synthesis may then be initiated using an oligonucleotide primer complementary to the linker.
- the enzyme T4 RNA ligase may be used to attach a donor 5' phosphate of a single-stranded DNA linker to the 3' end of RNA in an efficient reaction (see Uhlenbeck and Gumport, 1982).
- the primary function of the oligonucleotide linker is to provide an anchor sequence for binding of a first strand cDNA synthesis primer. Therefore, the precise nucieotide sequence of the oligonucleotide linker is generally not material to the invention.
- RNA strands containing analogue bases such as
- Br-U may be faithfully copied into first-strand cDNA using reverse transcriptase .
- copying of transcripts containing nucieotide analogues proceeds equally as efficiently as copying of ordinary unlabelled transcripts.
- the first-strand cDNAs derived from copying of nucieotide analogue labelled transcripts may also be converted to double-stranded cDNA using reverse transcriptase.
- RNA transcripts Once the labelled RNA transcripts have been converted to cDNA the resultant cDNA population may be analysed in order to determine the composition of the RNA population from which the cDNA was derived.
- cDNA is much more stable than RNA, i.e. less susceptible to degradation; and
- the pool of double-stranded cDNAs derived from the labelled RNA transcripts may be cloned into a suitable vector to form a library of cDNA clones.
- the vector will be one that facilitates direct sequencing of the cDNA inserts. It is then a matter of routine to sequence the inserts of each of the clones in the library. The resulting sequence data may be compared to the available databases in order to determine the numbers and types of cDNA clones present in the library.
- RNA transcripts When the starting material is labelled nascent RNA transcripts, the fragments of sequence derived from each of the clones are called 'nascent transcript tags' or NTTs, as opposed to expressed sequence tags or ESTs which are derived from poly (A) + RNA.
- cDNA libraries derived from nascent RNA are more complex than conventional libraries made from poly (A) + RNA, which yield ESTs. They contain cDNAs from all transcription units, and each transcription unit will have representatives with 3' ends differing in length by one nucieotide, as engaged polymerases will be found at every point along a transcription unit.
- NTTs the number of transcripts in a cDNA library derived from nascent transcripts
- a conventional library might contain 100 ESTs with the same 3' ends.
- some 3' termini will be in untranslated regions (at 5' and 3' ends, and in introns); no such termini would be found in conventional cDNA libraries.
- the analysis of NTTs therefore gives a snapshot of where all engaged RNA polymerases are located on the genome. It provides information on relative activity of all transcription units, and on where polymerases 'pause' within them.
- Direct sequencing has several advantages: it is straightforward, it uses an established approach, and it yields NTTs long enough to be identified uniquely in the genome. At one level it also provides redundancy - some NTTs will share sequences from the same transcription unit. But at another level, these NTTs derived from the same transcription unit will rarely have the same 3' ends.
- Fluorescent tags may be attached to cDNA copies of the labelled transcripts and the tagged cDNAs hybridized to 'microarrays' .
- the tagged cDNAs will most preferably be hybridized to arrays covering the complete human genome, including all transcription units with their introns, and 5' and 3' regions, as well as non-genic transcription units.
- SAGE The numbers and abundance of different messages in a cell - or cDNAs generated as above - can be catalogued rapidly using SAGE (Velculescu et al., 1995; Zhang et al., 1997; Kinzler et al . , US Patent No. 5,695,937).
- SAGE is based on two principles: First, short sequence tags of 9-15 nucleotides are generated that contain sufficient information to identify many transcripts; second, many transcript tags may be concentrated into one molecule that is sequenced using an automated sequencer, so that the identity of multiple tags can be uncovered in one sequencing run. The expression pattern of any population of transcripts can be evaluated quantitatively by determining the abundance of individual tags and identifying the gene corresponding to each tag.
- SAGE profiles for various cell types are now available, including those of cells from normal human pancreas, colorectal epithelium, and non-small cell lung cancer (eg Hibi et al., 1998; see also http: //www. ncbi .nlm. nih. gov/SAGE) .
- SAGE can be applied directly to the analysis of cDNAs derived from nascent RNA, as illustrated in Fig. 1 by way of example only.
- the reaction scheme illustrated in Figure 1 gives a maximum of 15 nucleotides/tag that can be used to screen databases.
- the present inventors have developed a modified SAGE technique which leads to the production of sequence tags from the extreme 3' ends of transcripts. This method, described in detail below, is particularly useful in the analysis of nascent RNA transcripts .
- RNA/cDNA populations which is similar to SAGE but which retains information on the 3 ' ends of nascent transcripts. Using this method, illustrated schematically by way of example only in Figure 3, many (sometimes overlapping) NTTs will be derived from each transcription unit so active transcription units can be identified uniquely (Fig. 2, bottom) .
- RNA transcripts which comprises:
- RNA transcripts ligating the 3' ends of the RNA transcripts to single-stranded oligonucleotide linkers comprising an enzyme recognition site that allows DNA cleavage at a site spaced a defined distance from the recognition site to form linked RNA;
- the above method is a modified SAGE technique which differs from conventional SAGE techniques known in the prior art in that it leads to the identification of short nucieotide sequence tags from the extreme 3' ends of RNA transcripts.
- This is achieved by ligating oligonucleotide linkers containing a recognition site for a tagging enzyme (i.e. an enzyme which cleaves at a defined distance from its recognition site) directly to the 3' ends of the RNA transcripts.
- a tagging enzyme i.e. an enzyme which cleaves at a defined distance from its recognition site
- a sample of RNA to be analysed is first divided into two pools of approximately equal size.
- the first of these RNA pools is ligated to a first oligonucleotide linker, the 5' ends of the linkers being joined to the 3' ends of the RNA transcripts, to form a first pool of linked transcripts.
- the second RNA pool is similarly ligated to a second oligonucleotide linker to form a second pool of linked transcripts.
- the ligation of a single-stranded DNA oligonucleotide linker to the 3' end of an RNA transcript may be carried out using the enzyme T4 RNA ligase, according to standard molecular biology protocols .
- the first and second oligonucleotide linkers both contain recognition sites for a tagging enzyme, i.e. a restriction enzyme which cuts at a defined distance downstream of its recognition site.
- the linkers should preferably contain a second restriction enzyme recognition site and most preferably the second restriction site will overlap with the recognition site for the tagging enzyme.
- the second restriction sites are added to facilitate concatenation of ditags, as described JD ⁇ IOW, and any convenient restriction sites may be used.
- Xmal sites are used in conjunction with BsmFI tagging enzyme sites.
- the first and second linkers are preferably DNA oligonucleotides and may be of identical nucieotide sequence or may have different sequences.
- linkers having identical sequences it is, of course, possible to carry out the method without first dividing the RNA into two separate pools. If linkers of different sequences are to be used then it is necessary to divide the RNA into two pools prior to the ligation of the linkers. The two pools of linked RNA may then be combined together for the subsequent steps of cDNA synthesis, cleavage with tagging enzyme and ligation to form ditags or may be kept separate up until the ligation step.
- linkers having identical sequences it is, of course, possible to carry out the method without first dividing the RNA into two separate pools. If linkers of different sequences are to be used then it is necessary to divide the RNA into two pools prior to the ligation of the linkers. The two pools of linked RNA may then be combined together for the subsequent steps of cDNA synthesis, cleavage with tagging enzyme and ligation to form ditags or may be kept separate up until the ligation step.
- the skilled person will readily appreciate that such variations may be made without departing from
- the linked RNAs are used as templates for synthesis of double-stranded cDNA.
- Synthesis of the first cDNA strand is primed by first-strand synthesis primers complementary to the oligonucleotide linkers.
- the first- strand synthesis primers are linked to a capture label which will allow specific capture of the cDNA strands.
- the first-strand synthesis primers are conjugated with a biotin capture label which allows capture of the cDNA via biotin/avidin or biotin/streptavidin binding. Other types of capture label may be used with equivalent effect.
- the double-stranded cDNAs are cut with the tagging enzyme which cuts a defined distance downstream of its recognition site in the linker sequence added to the 3' end of the template RNA.
- the most preferred tagging enzyme is BsmFI which cuts between -10 and -14 nucleotides away from its recognition sequence. Treatment with BsmFI releases cDNA fragments, each one containing the linker plus 10-14 nucleotides derived from the extreme 3' end of one primary transcript.
- BsmFI as the tagging enzyme.
- Other enzymes which share the characteristic of cutting a defined distance away from the recognition site may be used, in particular other type IIs restriction enzymes.
- other tagging enzymes may be used which lead to the generation of longer tags. In general, the longer the tags the easier it is to assign them to an unambiguous location within the genome .
- a purification or capture step may be included to separate the tags away from the cDNA fragments cleaved off by the tagging enzyme.
- a capture label is included in the primer used for first-strand cDNA synthesis the purification step may be easily carried out using a binding agent specific for the capture label. For example, if biotin is used as the capture label then labelled tags may be separated from unlabelled cDNA fragments using avidin or streptavidin coated beads. Once nucieotide tags have been generated from a defined position in the RNA transcripts and purified/captured, the tags are sequenced to provide an analysis of the original RNA transcripts.
- tags are ligated together to form ditags. If the tagging enzyme used to generate the tags was one which generates 3' or 5 ' overhangs, then the overhanging ends must be filled in to generate blunt ends prior to ligation.
- the 'filling in' reaction may be carried out using Klenow polymerase-a standard technique routinely used in molecular biology. Once the ends of the tags have been made blunt, the ligation reaction to form ditags may performed using a DNA ligase, according to standard molecular biology protocols.
- the amplification may be performed by conventional PCR using a pair of amplification primers corresponding to regions of the oligonucleotide linkers added to the 3' ends of the RNA transcripts at the start of the procedure.
- individual ditags may be concatenated into chains of ditags. This is achieved by first cleaving the ditags with a restriction enzyme which cleaves at restriction sites within the regions of the ditags derived from the single-stranded oligonucleotide linkers' (the second restriction sites described above) . If the pool of ditags has been subject to an amplification step then the amplification product is cut with the relevant enzyme. The cleaved ditags may then be concatenated using a conventional DNA ligation reaction.
- the concatamers of ditags are preferably cloned into a standard cloning vector and amplified by conventional PCR.
- the PCR products may then be sequenced directly and also sized by gel electrophoresis to provide an indication of the number of ditags present in the concatamer.
- the modified SAGE technique of the invention is of general applicability and may be used to characterize essentially any pool of RNAs isolated from any cell, cell population or tissue. It is particularly suitable for use in the analysis of nascent RNA transcripts. Therefore, in a preferred embodiment the invention provides a method for analysing the primary transcripts produced in a cell or cell population comprising the steps of: labelling nascent RNA transcripts transcribed in the cell or cell population with a nucieotide analogue; purifying the labelled transcripts; and analysing the labelled transcripts using the modified SAGE method according to the second aspect of the invention.
- the invention further provides a nucleic acid composition derived from a cell or cell population comprising at least one ditag, wherein each ditag comprises two covalently joined nucleic acid tags in opposite orientation, wherein each tag corresponds to the extreme 3' end of a primary RNA transcript expressed in said cell or cell population.
- the nucleic acid composition of the invention may be synthesised using the modified SAGE protocol of the invention, up to the step of ligating pairs of tags to form ditags, starting from a pool of nascent RNA isolated from a cell or cell population, for example by selective labelling and purification of nascent RNA as described in connection with the first aspect of the invention.
- the invention also encompasses nucleic acid compositions comprising concatenated ditags.
- the invention provides a method of detecting and characterising transcripts associated with a specific transcription unit within a pool of RNA, the method comprising steps of:
- a second round of PCR amplification using nested or hemi-nested PCR primers may be included between step (iii) and step (iv) is required.
- step (iv) Any of the many techniques known in the art for the detection of PCR amplification products may be used in step (iv) .
- specific sequences may be detected by probing a Southern blot of the PCR products with a probe corresponding to a region of the desired target sequence.
- the specific method of the invention is of general applicability to the detection and characterisation of specific transcripts from essentially any transcription unit amongst a population of RNA transcripts the method is particularly suitable for the detection and/or characterisation of specific transcripts within a population of nascent RNA.
- the invention provides a method of detecting and characterising primary transcripts associated with a specific transcription unit within a pool of nascent RNA derived from a given cell or cell type, the method comprising : labelling nascent RNA transcripts transcribed in the cell or cell population with a nucieotide analogue; purifying the labelled transcripts; and detecting and characterising transcripts associated with a specific transcription unit within the labelled transcripts using a method according to the third aspect of the invention.
- RNA polymerase Since the 3' ends of nascent transcripts are, by definition, highly variable depending on whereabouts in the gene the RNA polymerase is positioned at a particular point in time known methods such as RT-PCR, which relies on two primers specific for the transcript of interest, and conventional 3' RACE, which utilises a gene-specific primer and a primer complementary to the poly (A) + tail, are not suitable for use in characterising nascent RNA transcripts. In contrast, the specific method of the invention can be used for this purpose because of the step of ligating a linker to the 3' ends of the transcripts.
- Figure 1 illustrates a specific example of a method for analysing primary RNA transcripts according to the invention.
- Br-UTP is used as the nucieotide analogue and the double-stranded cDNAs derived from the labelled nascent RNA transcripts are characterised by conventional SAGE.
- Figure 2 illustrates the types of sequence tags which may be generated from four RNA transcripts of varying length using conventional SAGE (approach (i) ) or the modified SAGE method of the invention (approach (iii)) •
- Figure 3 illustrates a specific example of the modified SAGE method of the invention.
- Br-UTP is used as the labelled nucieotide analogue
- the cDNA synthesis primer is conjugated to a biotin capture label (bio)
- BsmFI is used as the tagging enzyme.
- Figure 4 illustrates a specific example of a method of detecting and characterising transcripts associated with a specific transcription unit according to the invention.
- This specific example includes a nested PCR step, the final amplification products being detected by Southern blotting.
- Figure 5 demonstrates that reverse transcriptase can copy Br-RNA, biotin-RNA and fluorescein-RNA into cDNA.
- Lane 1 Marker- ⁇ DNA cut with Hindl l l .
- Lanes 2,7 ATP, CTP, GTP, UTP.
- Lane 38 ATP, CTP, GTP, Br-UTP (Sigma).
- Lane 4,9 ATP, biotin-11-CTP (NEN) , GTP, UTP.
- Lane 510 ATP, CTP, GTP, fluorescein-12-UTP (NEN).
- Lane 611 ATP, CTP, GTP Lane 12 Marker-100 bp ladder
- the double-stranded DNA template ( ⁇ 4.4kb) is visible in lanes 2-11. Different amounts of RNA product are seen in lanes 2-5 and 7-9, but little (if any) RNA can be seen in lanes 6, 10, 11. Unincorporated fluorescein-UTP is visible at the bottom of lane 5, but this is removed by gel filtration (lane 10) .
- Lanes 1,12 Markers as above. Lanes 2,3: Normal RNA template (A, lane 7). Lanes 4,5: Br-RNA template (A, lane 8) . Lanes 6,7 Biotin-RNA template (A, lane 9) . Lanes 8,9: Fluorescein-RNA template (A, lane 10] Lanes 10,11 No RNA template (A, lane 11) .
- the arrow indicates a cDNA of ⁇ 1.8kb. All RNA templates give cDNA smears of ⁇ 1.8kb in the presence of reverse transcriptase (lanes 2,4,6,8). The bands at ⁇ 4.4kb (lanes 2,4,6) probably represent short labelled cDNAs hybridized to denatured DNA strands of -4.4kb.
- Example 1-to illustrate copying of transcripts containing nucieotide analogues into cDNA by reverse transcriptase
- RNA or Br-RNA with poly (A) at its 3' end was incubated ⁇ reverse transcriptase in the presence of oligo(dT) 15 , dATP, dGTP, dTTP, plus [ 32 P]dCTP.
- samples were denatured, and products separated from unincorporated nucleotides and primers. Copying by reverse transcriptase was monitored by sizing the resulting cDNA strands on an agarose gel.
- RNA and Br-RNA gave similar distributions of cDNA strands; these distributions extended in size up to ⁇ 1.8kb, the length of complete copies (Fig. 5B, lanes 2,4) and they were not seen when reverse transcriptase was omitted from the reaction (Fig. 5B, lanes 3,5). This result was confirmed by autoradiography (Fig. 5C, lanes 2-5) . As roughly the same amounts of RNA and Br- RNA were added to the reactions (Fig. 5A, lanes 7,8), and as the patterns of the resulting cDNA strands were roughly equivalent, reverse transcriptase must copy
- Br-RNA into cDNA yielded -50% as much cDNA as RNA (Fig. 5C, lanes 2,4).
- Br-RNA also supported incorporation of [ 32 P]dCTP (measured by scintillation counting) into acid-insoluble material at a rate (measured over 30 min) of 53% of that given by the natural RNA (not shown) .
- the fluorescein-RNA template supported incorporation of [ 32 P]dCTP into acid- insoluble material at a rate of 0.7% of that given by the natural RNA (not shown).
- oligo(dT) 15 will only prime cDNA synthesis using a full-length transcript that contains poly (A) at its 3' end, this incorporation - though slight - suggests that reverse transcriptase can copy fluorescein-RNA into cDNA reasonably efficiently (as so few full-length transcripts are present) .
- Reverse transcriptase would be expected to copy a Br-RNA (or fluorescein-RNA) template in which only some of the Us had been replaced by Br-U (or fluorescein-U) more efficiently than one in which every U had been replaced. Similarly, it would copy a biotin-RNA template in which only some Cs had been replaced by biotin-C more efficiently than one in which every C had been replaced.
- Partially- substituted RNA templates are generated when transcription is allowed to proceed in the presence of both UTP and Br-UTP, as is the case when cells are incubated in Br-U.
- Transcripts containing different nucieotide analogs were generated using the RiboMax Large Scale RNA Production System T7 (Promega). T7 polymerase was allowed to transcribe l ⁇ g of a linear template in the presence of the different nucieotide triphosphates indicated. The manufacturer's conditions were used, except that each NTP was present at a concentration of ImM. After the reaction, the template and resultant transcripts were purified free of unincorporated nucleotides by gel filtration on Sephacryl S-400HR (Pharmacia) in samples applied to lanes 7-11 of Fig. 5A. Samples (1 and 2 ⁇ l for unfiltered and filtered samples, respectively) were applied to 1.2% agarose, subjected to electrophoresis, stained with ethidium, and photographed under UV light (Fig. 5A) .
- Nucleic acids in the agarose gel were blotted on to a positively-charged nylon membrane (Amersham) and radioactively detected using a Phosphorimager (Fig. 5C) .
- RNA polymerases are localized within discrete transcription 'factories' in human nuclei. J. Cell Sci. 109, 1427-1436. Ishii, M., Hashimoto, Si, Tsutsumi, S., Wada, Y.,
- RNA polymerase II evidence for cotranscriptional splicing. Mol. Cell. Biol. 14, 7219-7225. Zhang, L., Zhou, W., Velculescu, V.E., Kern, S.E.,
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EP02700484A EP1364059A1 (en) | 2001-02-28 | 2002-02-27 | Methods for analysis of rna |
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DE4319468C1 (en) * | 1993-06-11 | 1994-11-17 | Mueller Manfred W | Method for producing a deoxyribonucleic acid (DNA) strand (cDNA strand) complementary to a ribonucleic acid (RNA) molecule, and the use of the method for the analysis of RNA molecules |
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JP2004526443A (en) | 2004-09-02 |
EP1364059A1 (en) | 2003-11-26 |
GB0104993D0 (en) | 2001-04-18 |
US20040142339A1 (en) | 2004-07-22 |
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